Candidate: Dr. Stephanie J. Murphy plans to establish a biomedical research career integrating neuroscience and specialization in comparative medicine with the clinical aspects of stroke. Her extensive formal doctoral training in biochemistry, clinical training in comparative medicine, and postdoctoral research in cerebral ischemia has given her a broad multidisciplinary background to achieve this goal. Environment: The A/CCM Research Division at the Johns Hopkins Medical Institutions offers exceptional personal guidance, interactive and experienced researchers, and excellent physical facilities with which to execute this research proposal. Drs. Patricia D. Hurn and Richard J. Traystman have offered their support and mentorship to Dr. Murphy. Research: We have shown that brain outcomes after experimental stroke are gender-specific and linked to reproductive hormone status. There are striking differences in post-ischemic brain injury after middle cerebral artery occlusion (MCAO) in female vs. male rat, suggesting that females enjoy substantial neuroprotection when confronted with an ischemic episode. Preliminary work suggests that reproductive steroids may reduce brain injury in older animals. The work proposed will clarify the neuroprotective importance of estrogen, alone or with progesterone, in reproductively senescent female (RSF) ischemic rat brain and assess if estrogen or combined hormone therapy alters ischemic outcome by reducing injury due to dopaminergic hyperactivity and hydroxyl radical formation. In Aim 1, we will determine if hormone replacement provides neuroprotection in RSF rats during reversible MCAO. Ischemic outcomes are determined by infarction volume analysis. Aim 2 determines if estrogen or combined hormone treatment attenuates ischemic injury by reducing dopaminergic neurotransmission utilizing microdialysis. Aim 3 determines if reproductive steroids alter infarction outcomes after MCAO in RSF rats by reducing hydroxyl radical formation from either dopamine (DA)-induced or non-DA derived sources as determined by salicylate trapping and microdialysis. In Aim 4, we will assess if ovarian steroids induce DA-modulated neuroprotection through increases in DA uptake and intracellular transport via increased expression of the DA membrane transporter (DAT) and the vesicular monoamine transporter (VMAT2), respectively. DAT and VMAT2 expression are determined using in situ hybridization, Northern blots, RNAase protection assays, immunohistochemistry, and Western blots.